Return to study ST000916 main page
MB Sample ID: SA053927
| Local Sample ID: | NASH035 |
| Subject ID: | SU000954 |
| Subject Type: | Human clinical study |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 23-83 |
| Gender: | Male and Female |
| Human Ethnicity: | Mixed |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000954 |
| Subject Type: | Human clinical study |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 23-83 |
| Gender: | Male and Female |
| Human Ethnicity: | Mixed |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| NASH035 | SA053927 | FL010087 | NASH | Diagnosis |
Collection:
| Collection ID: | CO000948 |
| Collection Summary: | Human samples were collected according to a protocol approved by Vanderbilt University Medical Center's Internal Review Board (#120829) and under informed written patients' consent prior to inclusion in this study. Sample sizes were selected to minimize the invasive procedures. Plasma samples were obtained from patients' blood collected during standard of care surgical procedures. Urine samples were collected from patients' Foley catheters placed for standard of care procedure. Liver samples were obtained from the excess tissue collected as part of the standard of care liver biopsies performed at the time of surgery that would otherwise be discarded. Subsequently, studies at University of California, San Diego were conducted under further auspices of University of California, San Diego Internal Review Board #121220. |
| Sample Type: | Plasma |
Treatment:
| Treatment ID: | TR000968 |
| Treatment Summary: | - |
Sample Preparation:
| Sampleprep ID: | SP000961 |
| Sampleprep Summary: | Plasma sample extraction. One hundred microliters of thawed plasma containing 4 µl of internal standard mix were extracted with 500 µl cold (-20°C) 70% CH3OH by incubation in an ice bath for 30 min. After mixing by vortex at 4°C for 1 min and centrifugation (4°C, 18,000 g, 10 min), the solvent was evaporated from the supernatant. The residue was dissolved in 200 µl of resuspension solvent, vortexed to mix (1 min at 4°C), and centrifuged (4°C, 18,000 g, 10 min) to remove any insoluble material. GPLs: GPLs from liver samples were extracted and analyzed by MS essentially as described in (20, 21). Extraction and analysis of plasma samples was according to previously published procedures (22). Cardiolipin, coenzyme Q, and dolichol: Lipid extractions were performed based on the Bligh and Dyer method with minor modifications (23-25). FAs and eicosanoids: FFAs were extracted essentially as previously described after supplementation with deuterated internal standards (Cayman Chemicals) (26, 27). Eicosanoids were isolated via solid phase extraction, utilizing 25 deuterated internal standards (28, 29). Sterols and oxysterols: Sterols and oxysterols were extracted using previously described methods (30). Neutral lipids: Cholesteryl esters (CEs), TAGs, and DAGs were extracted from weighed liver tissue (0.5-1 mg) suspended in 0.5 ml PBS that had been homogenized by sonication. Extractions of plasma (0.05 ml diluted to 0.1 ml with PBS), urine (1 ml), and tissue sonicates were carried out using 1 ml hexane:methyl t-butyl ether (1:1, v/v), essentially as previously described (31). Sphingolipids: Sphingolipids from liver, plasma, and urine were extracted following previously published procedures (32, 33), with the exception that methylene chloride was substituted for chloroform for the single-phase extraction of sphingoid bases. 20. Ivanova P. T., Milne S. B., Byrne M. O., Xiang Y., Brown H. A. 2007. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Methods Enzymol. 432: 21-57. 21. Myers D. S., Ivanova P. T., Milne S. B., Brown H. A. 2011. Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation. Biochim. Biophys. Acta. 1811: 748-757. 22. Quehenberger O., Armando A. M., Brown H. A., Milne S. B., Myers D. S., Merrill A. H., Jr, Bandyopadhyay S., Jones K. N., Kelly S., Shaner R. L., et al. 2010. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 51: 3299-3305. 23. Guan Z., Li S., Smith D., Shaw W., Raetz C. 2007. Identification of N-acylphosphatidylserine molecules in eukaryotic cells. Biochemistry. 46: 14500-14513. 24. Tan B. K., Bogdanov M., Zhao J., Dowhan W., Raetz C. R., Guan Z. 2012. Discovery of cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates. Proc. Natl. Acad. Sci. USA. 109: 16504-16509. 25. Wen R., Lam B., Guan Z. 2013. Aberrant dolichol chain lengths as biomarkers for retinitis pigmentosa caused by impaired dolichol biosynthesis. J. Lipid Res. 54: 3516-3522. 26. Quehenberger O., Armando A., Dumlao D., Stephens D. L., Dennis E. A. 2008. Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot. Essent. Fatty Acids. 79: 123-129. 27. Quehenberger O., Armando A. M., Dennis E. A. 2011. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim. Biophys. Acta. 1811: 648-656. 28. Deems R., Buczynski M. W., Bowers-Gentry R., Harkewicz R., Dennis E. A. 2007. Detection and quantitation of eicosanoids via high performance liquid chromatography-electrospray ionization-mass spectrometry. Methods Enzymol. 432: 59-82. 29. Dumlao D. S., Buczynski M. W., Norris P. C., Harkewicz R., Dennis E. A. 2011. High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim. Biophys. Acta. 1811: 724-736. 30. McDonald J. G., Smith D. D., Stiles A. R., Russell D. W. 2012. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53: 1399-1409. 31. Hutchins P. M., Barkley R. M., Murphy R. C. 2008. Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J. Lipid Res. 49: 804-813. 32. Shaner R. L., Allegood J. C., Park H., Wang E., Kelly S., Haynes C. A., Sullards M. C., Merrill A. H., Jr 2009. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J. Lipid Res. 50: 1692-1707. 33. Sullards M. C., Liu Y., Chen Y., Merrill A. H., Jr 2011. Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Biochim. Biophys. Acta. 1811: 838-853. |
Combined analysis:
| Analysis ID | AN001491 | AN001492 | AN001493 | AN001494 | AN001495 | AN001496 |
|---|---|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS | MS | MS |
| Chromatography type | Unspecified | Unspecified | Unspecified | Unspecified | Unspecified | Unspecified |
| Chromatography system | Multiple | Multiple | Multiple | Multiple | Multiple | Multiple |
| Column | Multiple | Multiple | Multiple | Multiple | Multiple | Multiple |
| MS Type | Other | Other | Other | Other | Other | Other |
| MS instrument type | - | - | - | - | - | - |
| MS instrument name | ||||||
| Ion Mode | UNSPECIFIED | UNSPECIFIED | UNSPECIFIED | UNSPECIFIED | UNSPECIFIED | UNSPECIFIED |
| Units | pmol/ml | nmol/ml | pmol/ml | nmol/ml | pmol/ml | pmol/ml |
Chromatography:
| Chromatography ID: | CH001048 |
| Chromatography Summary: | FFAs were analyzed by stable isotope dilution GC-MS after derivatization, essentially as described previously (26, 27). This method quantifies 33 FAs including all major and minor saturated FAs, monounsaturated FAs, and PUFAs containing 12 to 26 carbons. Eicosanoids were analyzed by a stable isotope dilution LC/MS method utilizing 26 deuterated internal standards (28, 29). The metabolites were quantified after separation by reverse phase chromatography on a 2.1 × 100 mm BEH Shield column, 1.7 µM (Waters, Milford, MA) employing an Acquity UPLC system (Waters). Detection and quantification were performed on an AB SCIEX 6500 QTrap mass spectrometer equipped with an IonDrive Turbo V source (AB SCIEX, Framingham, MA), operated in negative ionization mode via MRM, using standard curves generated from 145 authentic quantification standards (34). The method analyzes an additional 13 metabolites based on authentic primary standards, but which cannot be quantified due to the lack of appropriate internal standards. Data analysis was performed using MultiQuant 2.1 software (AB SCIEX). 26. Quehenberger O., Armando A., Dumlao D., Stephens D. L., Dennis E. A. 2008. Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot. Essent. Fatty Acids. 79: 123-129. 27. Quehenberger O., Armando A. M., Dennis E. A. 2011. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim. Biophys. Acta. 1811: 648-656. |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
| Chromatography ID: | CH001049 |
| Chromatography Summary: | Sterols and oxysterols were measured using methods previously described (30). Plasma total cholesterol was measured using a Vitros 250 chemistry system (Ortho-Clinical Diagnostics, Rochester, NY). Plasma free cholesterol and liver free and total cholesterol were measured using methods adapted from (30). 30. McDonald J. G., Smith D. D., Stiles A. R., Russell D. W. 2012. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53: 1399-1409. |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
| Chromatography ID: | CH001050 |
| Chromatography Summary: | Cardiolipin analysis was achieved with normal phase LC coupled with high-resolution MS performed on a TripleTOF 5600 system (AB SCIEX, Foster City, CA) (24). Dolichol and coenzyme Q were analyzed by reverse phase LC coupled with multiple reaction monitoring (MRM) MS utilizing a 4000 Q-Trap hybrid triple quadrupole linear ion-trap mass spectrometer (AB SCIEX) (22, 23). For dolichol analysis, MRM was performed in negative ion mode, with the precursor ions being the (M+acetate)- adduct ions and the product ions being the acetate ions (m/z 59). For coenzyme Q analysis, MRM was carried out in positive ion mode, with ammonium adducts (M+NH4)+ as precursor ions and the proton adducts of the quinone ring of coenzyme Q (m/z 197) as product ions. For quantitation, an internal standard mixture composed of a cardiolipin mix (Avanti Polar Lipids, Inc.), nor-dolichol (13-22) (Avanti Polar Lipids, Inc.), and yeast coenzyme Q6 (Sigma) was added during the first step of lipid extraction (22). 22. Quehenberger O., Armando A. M., Brown H. A., Milne S. B., Myers D. S., Merrill A. H., Jr, Bandyopadhyay S., Jones K. N., Kelly S., Shaner R. L., et al. 2010. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 51: 3299-3305. 23. Guan Z., Li S., Smith D., Shaw W., Raetz C. 2007. Identification of N-acylphosphatidylserine molecules in eukaryotic cells. Biochemistry. 46: 14500-14513. 24. Tan B. K., Bogdanov M., Zhao J., Dowhan W., Raetz C. R., Guan Z. 2012. Discovery of cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates. Proc. Natl. Acad. Sci. USA. 109: 16504-16509. |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
| Chromatography ID: | CH001051 |
| Chromatography Summary: | The organic solvent extraction layer containing CEs, TAGs, and DAGs was taken to dryness, then derivatized with 2,5-difluorophenylisocyanate to convert DAGs to urethane derivatives (35). The derivatized extract was separated by normal phase LC as previously described (35). The CEs eluting first from the LC column were detected by 20 specific MRM transitions corresponding to each [M+NH4]+ ion being collisionally activated to m/z 369.3. During elution of TAGs, the mass spectrometer was set to carry out full mass scanning from m/z 400-1,000. The DAGs were detected by neutral loss scanning of 190 Da. 35. Leiker T. J., Barkley R. M., Murphy R. C. 2011. Analysis of diacylglycerol molecular species in cellular lipid extracts by normal-phase LC-electrospray mass spectrometry. Int. J. Mass Spectrom. 305: 103-109 |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
| Chromatography ID: | CH001052 |
| Chromatography Summary: | Sphingolipids were analyzed by LC-MS/MS essentially as described in (32, 33) with minor modifications to include the 1-deoxy-sphingolipids as generally described in (36). 32. Shaner R. L., Allegood J. C., Park H., Wang E., Kelly S., Haynes C. A., Sullards M. C., Merrill A. H., Jr 2009. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J. Lipid Res. 50: 1692-1707. 33. Sullards M. C., Liu Y., Chen Y., Merrill A. H., Jr 2011. Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Biochim. Biophys. Acta. 1811: 838-853. 36. Zitomer N. C., Mitchell T., Voss K. A., Bondy G. S., Pruett S. T., Garnier-Amblard E. C., Liebeskind L. S., Park H., Wang E., Sullards M. C., et al. 2009. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals. J. Biol. Chem. 284: 4786-4795. |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
| Chromatography ID: | CH001053 |
| Chromatography Summary: | Extracted GPLs from liver and plasma were analyzed by MS, as described elsewhere (20, 21). 20. Ivanova P. T., Milne S. B., Byrne M. O., Xiang Y., Brown H. A. 2007. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Methods Enzymol. 432: 21-57. 21. Myers D. S., Ivanova P. T., Milne S. B., Brown H. A. 2011. Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation. Biochim. Biophys. Acta. 1811: 748-757. |
| Instrument Name: | Multiple |
| Column Name: | Multiple |
| Chromatography Type: | Unspecified |
MS:
| MS ID: | MS001374 |
| Analysis ID: | AN001491 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS001375 |
| Analysis ID: | AN001492 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS001376 |
| Analysis ID: | AN001493 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS001377 |
| Analysis ID: | AN001494 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS001378 |
| Analysis ID: | AN001495 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS001379 |
| Analysis ID: | AN001496 |
| Instrument Type: | - |
| MS Type: | Other |
| Ion Mode: | UNSPECIFIED |
